Since 1914, the safety of our water supply has generally been protected by the use of assays which detect the growth of certain types of bacteria, commonly referred to as indicator bacteria, to infer the presence of pathogens. Indicator bacteria, such as fecal coliforms and total coliforms, are found in fecal matter along with many pathogens. Although many indicator bacteria do not cause disease in humans, their presence indicates a potential risk of exposure to pathogens. As a result, water in which such bacteria are found will be declared unsafe for human contact therewith. Pathogens are not routinely assayed by direct methods due to difficulty in their isolation and detection. In contrast, indicator bacteria are cultured in 24 to 48 hours and can be detected visually. The main disadvantage with this detection method is that the results do not indicate the present water quality. In addition, indicator assays fail to accurately assess the infectivity of water. The risk is overestimated in environments which stimulate the growth of the non-pathogenic indicator microorganisms (i.e. warm, nutrient-rich waters). Conversely, certain waterborne pathogens (i.e. Legionella and Naegleria fowleri) are not transmitted through the feces and thus are not associated with fecal indicator organisms. Even in situations in which pathogens and indicator bacteria are from the same fecal source, the indicator bacteria may be killed more quickly than hardier pathogens such as protozoan cysts, viruses or bacterial spores. These and other problems could be eliminated by using assays which directly detect and quantitate waterborne pathogens.
More recently, the polymerase chain reaction (PCR), has been used in the detection and classification of various microorganisms. While DNA hybridization is useful in some applications, it carries the distinct disadvantage of having a high detection limit (low sensitivity). PCR, on the other hand, eliminates the need to culture the microorganism and is extremely sensitive--capable of detecting a single cell. The first and most critical step in both methods is, of course, the isolation of DNA of sufficient purity for analysis.
Several methods exist for the isolation of DNA from bacterial cells. These methods essentially utilize the same basic procedure. Bacterial cells are lysed enzymatically (i.e., lysozyme treatment), mechanically (i.e., bead homogenization) or by repeated freeze-thaw cycles, or combinations of these, followed by dissolution of the cell membrane with alkali and detergents such as sodium dodecyl sulfate (SDS) (Maniatis et al., 1989; Tsai et al., Appl. Environ. Microbiol., 57:1070-1074, 1991; Bej et al., Appl. Environ. Microbiol., 57:1013-1017, 1991). The cell lysate is then treated with proteinases and hexadecyltrimethyl ammonium bromide (CTAB) to degrade proteins and precipitate carbohydrates, respectively. The most common proteinase used in this procedure is proteinase K. Finally, DNA is purified by extraction with phenol, chloroform and isoamyl alcohol. Variations of this basic method have been used to isolate DNA from soils, sediments and water samples for use in hybridization and PCR analysis (Somerville et al., Appl. Environ. Microbiol., 55, 548-554, 1989; Tsai et al., Appl. Environ. Microbiol., 59:353-357, 1993; Bej et al., Appl. Environ. Microbiol., 56:307-314, 1990). Although these methods can result in DNA of sufficient purity for both hybridization and PCR analysis, they are time consuming and involve expensive and toxic reagents. Further, the DNA obtained from soil and sediment samples is often of questionable purity and its analysis requires several days.
A substance in a supercritical fluid state is defined when it is above the critical temperature (the temperature above which the gas cannot be liquified no matter how high the pressure), and above the critical pressure (the pressure which will liquefy the gas at its critical temperature). At this point, the fluid has equal coexisting densities of its gaseous and liquid phases (Lange's Handbook of Chemistry, 13th ed, Dean, J. A., ed., McGraw-Hill, New York). The supercritical fluid is a viscous gas with properties analogous to those of liquid solvents (Hawthorne, Anal. Chem., 62:633-642, 1990). The difference between liquid solvents and supercritical fluids is that the solvent strength of a supercritical fluid can be controlled by changes in temperature and/or pressure. The most commonly used supercritical fluid is carbon dioxide (CO.sub.2) which is inert, nontoxic, nonflammable, inexpensive and available in a very pure form. CO.sub.2 has a low critical temperature (31.1.degree. C.) and critical pressure (72.85 atm).
Supercritical CO.sub.2 has been used to extract a variety of nonpolar compounds from both biological and non-biological sources (Lin et al., Biotechnol. Prog., 8:458-461, 1992). It has been used to extract alkanes, sulfur compounds, PCBs, pesticides and polycyclic aromatic hydrocarbons from soil and sediments (Hawthorne, ibid.; Hopfgartner et al., Org. Geochem., 15:397-402, 1990), as well as fatty acid and sterol lipid biomarkers from plant tissue, sediments, and filtered water samples (Klink et al., Org. Geochem., 21:437-441, 1994).
The number of viable microorganisms decreases after treatment with supercritical fluids. For example, cell inactivation of Saccharomyces cerevisiae increases with an increase in pressure at temperatures of 25-45.degree. C. and pressures of 68-204 atm (Lin et al., ibid.). Under these conditions, inactivation occurred in greater than 15 minutes at 25.degree. C. and 5 minutes at 35.degree. C. Increases in pressure or exposure time were correlated with an increase in adverse effects, including microbial death (Hoover et al., Food Technol., 43:99-107, 1989). When exposed to pressures of 300 to 450 atm, Pseudomonas exhibited morphological changes including cellular elongation, separation of the cell wall from the plasma membrane and clear areas of spongy or reticular structures in the cytoplasm (Hoover et al., ibid.; Kriss et al., Mikrobiolgiya, 38:88, 1969).
DNA appears very resistant to hydrostatic pressure. Structural integrity of calf thymus or salmon sperm DNA remained unchanged when pressures of up to 10,000 atm were applied for 60 min at 25-40.degree. C.
The present invention provides an apparatus and method for the rapid isolation of DNA of high purity from microorganisms present in environmental samples including water, soil and sediments. Importantly, this method can be used to detect the presence of pathogenic microorganisms in water supplies.